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Galactooligosaccharides (GOS) are an important class of dietary prebiotics that exert beneficial effects on intestinal microbiota and gut barrier function. In this study, high-purity GOS (HP-GOS) were investigated in vitro and in vivo and confirmed as prebiotic ingredients in rat diet. HP-GOS were successfully produced using a two-step process, enzymatic hydrolysis and fermentation by yeast. They were found to serve as a good substrate and carbon source for supporting the growth of probiotic bacteria more effectively than other commercial GOS. Following administration of 1 % (by mass) of HP-GOS to rats, the growth of Bifidobacterium bifidum and B. longum in the gut increased most rapidly up to 12 h, and thereafter the increase was slow. Therefore, 1 % HP-GOS was found to be acceptable for the growth of probiotic bacteria. Groups of animals that were orally administered HP-GOS and bifidobacteria during the study, and the group administered HP-GOS during the 2nd (days 13–15) and 4th (days 28–30) period of the study had significantly (p<0.05) higher numbers of bifidobacteria in faeces than groups receiving a single dose of bifidobacteria. HP-GOS affected the expression of genes encoding glucagon-like peptide-1 (GLP-1) and peptide YY (PYY). There was a significant upregulation of GLP-1 and PYY mRNA with HP-GOS and bifidobacteria intake. We propose that the prebiotic properties of HP-GOS are potentially valuable for the production of functional foods for human consumption., Galaktooligosaharidi su važna klasa prebiotika koji blagotvorno djeluju na mikroorganizme probavnog trakta i time štite stijenke probavnih organa. U radu je ispitan in vitro i in vivo prebiotički učinak pročišćenih galaktooligosaharida, dodanih hrani za štakore. Pročišćeni galaktooligosaharidi uspješno su proizvedeni enzimskom hidrolizom i fermentacijom s pomoću kvasca. U usporedbi s galaktooligosaharidima dostupnim na tržištu, znatno su bolji izvor ugljika te podloga za rast probiotičkih bakterija. Nakon dodatka 1 % masenog udjela pročišćenih galaktooligosaharida hrani, bitno se povećao rast bakterija Bifidobacterium bifidum i B. longum u probavnom traktu štakora tijekom 12 sati, nakon čega je rast bio nešto sporiji. Zaključeno je da ta doza galaktooligosaharida ima prebiotički učinak. Skupine životinja dohranjivane pročišćenim galaktooligosaharidima i bifidobakterijama te skupina dohranjena galaktooligosaharidima nakon 13-15 i 28-30 dana uzgoja imale su bitno veći (p˂0,05) broj bakterija u izmetu od skupina dohranjenih samo jednom dozom bifidobakterija. Pročišćeni galaktooligosaharidi utjecali su na ekspresiju gena koji kodiraju GLP-1 (peptid sličan glukagonu) i peptid YY (PYY). Nakon dohrane prebioticima i bifidobakterijama došlo je do pojačane uzvodne transkripcije mRNA u genima za GLP-1 i PYY. Stoga smatramo da se pročišćeni galaktooligosaharidi zbog dragocjenih prebiotičkih svojstava mogu upotrijebiti u proizvodnji funkcionalne hrane.